The recent explosion in understanding vascular effects of gonadal steroids includes identification of two types of estrogen receptor (ER) and both genomic and non-genomic mechanisms. Still, major questions remain concerning which receptors and which mechanisms contribute most to the actions of gonadal hormones. Therefore, we will continue to integrate both functional and biochemical approaches, using chronic hormone treatment in vivo to emphasize physiologically relevant effects in the cerebral circulation. The major hypothesis is: Estrogen and testosterone alter cerebrovascular function by influencing endothelial mechanisms. Cerebral arteries will be isolated from rats and mice chronically treated with gonadal steroids. Vascular contractility, endothelial calcium, smooth muscle membrane potential, synthetic proteins for vasoactive factors and their release will be quantified. Four specific hypotheses will be tested: 1. Estrogen, by activating ERalpha, increases endothelial-dependent cerebrovascular dilation. Estrogen may influence at least three vasodilators: NO, prostacyclin and endothelial derived hyperpolarizing factor (EDHF). We will test whether estrogen treatment increases vasodilation mediated by EDHF, whether there are significant interactions among endothelial factors that modulate estrogen effects, whether estrogen treatment increases endothelial calcium, and whether effects of estrogen on cerebrovascular endothelial function are mediated by genomic actions of ERalpha. 2. Testosterone increases cerebrovascular constriction through endothelial-dependent mechanisms. Since testosterone treatment increases cerebrovascular constriction, we will test whether testosterone affects endothelial vasoconstrictor and/or vasodilator factors. 3. Induction of COX-2 or iNOS will be altered by estrogen or testosterone. We will monitor iNOS and COX-2 induction and determine if gonadal steroids modulate inflammatory responses. 4. Progestins will modulate effects of estrogen. We will assess whether chronic progestin treatment alters vascular effects of estrogen. Given known sex differences in incidence of cardiovascular disease as well as the applicability of hormone replacement therapy to a large segment of the population, this work has tremendous significance for understanding physiological control of the cerebral circulation as well as improvement of hormonal therapy.